ABSTRACT Immunotherapy is an effective treatment for certain cancer patients. Checkpoint blockade strategies using mAbs directed at CTLA-4 and/or PD1 facilitate CD8 cytotoxic T lymphocyte (CTL)-mediated destruction of tumors, leading to long-term disease stabilization and even remission in some patients with otherwise incurable disease. However, responders are restricted to a minority of patients who have endogenous CD8 T cells that target their own tumors. Moreover, only limited histological cancer types are blockade responsive. The precise specificities of those CTLs are largely unknown. A systematic vaccine elicitation strategy is required to benefit the majority of patients but a physical detection technique suitable for the challenge of identifying a handful of tumor antigens arising by non-synonymous mutation among 100,000 normal self- peptides bound to the same MHC molecule type is lacking. As a result, genomic or transcriptomic sequencing methods and indirect reverse immunology efforts are used to infer their identity on the tumor cell surface. Unfortunately, such standard T cell function-based methods fail to identify stealth (i.e. non-immunogenic) epitopes arrayed but unrecognized by the natural immune response (false negatives) while yielding many false positive results due to T cell crossreactivities. In Aim 1, we propose to develop and deploy an ultrasensitive Poisson detection liquid chromatography-data independent acquisition (LC-DIA) mass spectrometry (MS) method for antigen discovery to electronically capture the entire immune peptidome from small numbers of tumor cells (106) retrieved by clinical needle biopsy. In so doing, we can unambiguously identify the relevant epitopes as a focus for vaccine targeting. Our recent studies have shown that the T cell receptor (TCR) is an anisotropic mechanosensor whose exquisite specificity for an antigenic peptide bound to an MHC molecule (pMHC) and sensitivity (several copies per target cell) is revealed by piconewton force application as occurs during immune surveillance in vivo. Yet clinical analysis of human T cell responses is carried out in the absence of force, using high concentrations of peptide (micromolar) for in vitro stimulation that further minimizes specificity. Thus, in Aim 2, we shall develop massively parallel optical tweezer arrays to assess pMHC-TCR bond quality under force using individual T cells from tumors, tissues and/or blood that naturally arise in individuals or are induced through vaccination; conjoint single-cell RNA retrieval for TCR identification and bond lifetime will be incorporated into this platform. In Aim 3, the importance for protective CTL immunity of precise epitope detection as well as optimal single TCR-pMHC bond lifetime under load shall be proven using in vivo pre-clinical models of viral infection and tumor immunotherapy. These advances have the potential to revolutionize CD8 vaccine development by incisively defining the limited number of targets among the expansive immune peptidome with which to program an effective CTL response.